Variable region of the monoclonal antibody against the HBV S-surface antigen and a gene encoding the same

ABSTRACT

The present invention relates to a variable region of the monoclonal antibody against the S-surface antigen of hepatis B virus and a gene encoding the same, a recombinant vector containing the said gene, and a transformant obtained from the said recombinant vector.

This application is a divisional of U.S. Ser. No. 09/865,483, filed May29, 2001, now U.S. Pat. No. 6,680,053, the entire contents of which areherein incorporated by reference and for which priority is claimed under35 U.S.C. §120; and this application claims priority to Application No.2000-28938 filed in Korea on May 29, 2000 under 35 U.S.C. §119, thecontents of which are herein incorporated by reference.

TECHNICAL FIELD

The present invention relates to a variable region of the monoclonalantibody against S-surface antigen of hepatitis B virus and a geneencoding the same, a recombinant vector containing the said gene, and atransformant obtained from the said recombinant vector.

BACKGROUND ART

Hepatitis B virus (hereinafter, referred to as “HBV”), known as the Daneparticle, has a spherical feature of 42 nm diameter. The outer envelopecontains a large amount of hepatitis B surface antigens and surroundsthe inner nucleocapsid composed of 180 hepatitis B core proteins. Thenucleocapsid contains HBV genome, polymerase, etc (Summers et al., Proc.Nat. Acad. Sci, 72, 4579, 1975; Pierre Tiollais et al., Science, 213,406–411, 1981).

Within the HBV genome, the coding region of HBV surface antigenscontains three open reading frame start sites which share a commontermination codon producing same S domain. Thus, the HBV surfaceantigens may be classified into three types, i.e., (1) Small HBV SurfaceAntigen (hereinafter, referred to as “S-surface antigen”), containingonly the S domain, (2) Middle HBV Surface Antigen (hereinafter, referredto as “M-surface antigen”), containing the S domain and an additional 55amino acid domain known as Pre-S2, and (3) Large HBV Surface Antigen(hereinafter, referred to as “L-surface antigen”), containing the Pre-S1domain as well as the Pre-S2 and S domain. Among the expressed surfaceantigens, S-surface antigen is about 80% or more.

Subtypes of S-surface antigen were classified according to theirproperties of antibody recognition. Antigenic domains expressed in allsurface antigen were classified as determinant a. The four othersubtypes are d or y and w or r. Determinant d has a lysine at residue122 while y has arginine. Similarly, determinant w has a lysine atresidue 160 while r has arginine (Kennedy R. C. et al., J. Immunol. 130,385, 1983). Thus, serological types can be classified into foursubtypes, such as adr, adw, ayr and ayw (Peterson et al., J. Biol. Chem.257, 10414, 1982; Lars O. Marnius et al., Intervirology, 38, 24–34,1995).

The S-surface antigen specifically binds to hepatocyte (Leenders et al.,Hepatology, 12, 141, 1990; Irina Ionescu-Matiu et al., J. Med. Virology,6, 175–178, 1980; Swan N. T. et al., Gastroenterology 80, 260–264, 1981;Swan N. T. et al., Gastroenterology, 85, 466–468, 1983; Marie, L. M. etal., Proc. Nat. Acad. Sci. 81, 7708–7712, 1984). And, it has beenidentified that human hepatic plasma membrane contains target proteinssuch as apolipoprotein H and endonexin II which specifically bind toS-surface antigen (Mehdi H. et al., J. Virol., 68, 2415, 1994.; HertogsK. et al., Virology, 197, 265, 1993).

Meanwhile, in developing an useful therapeutic monoclonal antibody, ahumanized antibody is preferable because monoclonal antibodies obtainedfrom mice could cause an immune response when applying to human.

The Korean patent publication No. 1999-8650 has recently disclosed avariable region of the monoclonal antibody against a Pre-S1 epitopewhich solely exists in a L-surface antigen among the three HBV surfaceantigens (S-, M-, and L-surface antigens), a gene encoding the same, anda humanized antibody using the same. Because the L-surface antigen isonly 1˜2% of the expressed surface antigens, however, the L-surfaceantigen is inappropriate as a target for anti-HBV antibody developmentfor diagnostic as well as therapeutic purposes.

DISCLOSURE OF THE INVENTION

Accordingly, a primary object of the present invention is to provide agene encoding the variable region of a monoclonal antibody, specificallyrecognizing the S-surface antigen, especially determinant a, whichcommonly exists in all of the HBV surface antigens.

It is another object of the present invention to provide a recombinantvector comprising the above gene.

It is a further object of the present invention to provide atransformant obtained using the above recombinant vector.

It is still another object of the present invention to provide avariable region of the above monoclonal antibody.

In accordance with one aspect of the present invention, provided is agene encoding the monoclonal antibody variable region which specificallyrecognizes the HBV S-surface antigen.

The present inventors immunize mice with the determinant adr type ofS-surface antigen (International Enzymes Inc., USA) which is mostfrequently found in Korean HBV patients. The spleen cells obtained fromthe immunized mice were fused with myeloma cells (SP2O-Ag14, ATCCCRL-1581) to generate a large number of hybridoma cells which, followingsubsequent cloning and selection procedures, eventually give rise tonumerous monoclonal antibodies. The present inventors selectedmonoclonal antibodies specifically binding to the determinant a amongthe numerous monoclonal antibodies; and as a result, obtained ahybridoma cell line (A9-11-5) producing a distinct monoclonal antibodywhich specifically binds to the determinant a with high bindingaffinity.

The present inventors isolated total RNAs from the said hybridoma cellline to synthesize the cDNAs of light and heavy chains, and finally,obtained about 440 bp of light chain cDNA gene comprising SEQ ID NO. 5and about 460 bp of heavy chain cDNA gene comprising SEQ ID NO. 6,respectively.

From the said monoclonal antibody light and heavy chains, the CDR(complementarity determining region) residues were detected. As aresult, it is identified that the CDR residues of the light chain existat the positions of 23–36, 52–58, and 91–98 representing the peptides ofSEQ ID Nos. 9, 10, and 11, respectively. Further, it is found that theCDR residues of the heavy chain exist at the positions of 31–35, 50–65,and 98–103 representing the peptides of SEQ ID Nos. 12, 13, and 14,respectively.

Accordingly, the present invention includes, within its scope, a cDNAencoding a light chain variable region of a monoclonal antibody againsta HBV S-surface antigen, the said light chain variable region comprisingthe peptides of SEQ ID Nos. 9, 10, and 11. Further, the presentinvention includes a cDNA wherein the light chain variable region hasthe amino acid sequence of SEQ ID NO. 7, and preferably, a cDNAcomprising the nucleotide sequence of SEQ ID NO. 5.

And also, the present invention includes, within its scope, a cDNAencoding a heavy chain variable region of a monoclonal antibody againstthe HBV S-surface antigen, the said heavy chain variable regioncomprising the peptides of SEQ ID Nos. 12, 13, and 14. Further, thepresent invention includes a cDNA wherein the heavy chain variableregion has the amino acid sequence of SEQ ID NO. 8, and preferably, acDNA comprising the nucleotide sequence of SEQ ID NO. 6.

The above cDNA genes encoding the light or heavy chain variable regionof a monoclonal antibody may be inserted into plasmid vector such aspCRII (Invitrogen Co. USA) to give recombinant vectors. Accordingly, thepresent invention includes, within its scope, a recombinant vectorpCRA9Lv comprising the above cDNA encoding a light chain variable regionand a recombinant vector pCRA9Hv comprising the above cDNA encoding aheavy chain variable region.

Further, microorganisms, such as E. coli, may be transformed with theabove recombinant vectors, pCRA9Lv and/or pCRA9Hv, to obtaintransformants. Accordingly, the present invention includes atransformant E. coli DH5α/YRC-pCRA9Lv (KCTC 1011BP) and a transformantE. coli DH5α/YRC-pCRA9Hv (KCTC 1010BP) which are transformed with arecombinant vector pCRA9Lv and pCRA9Hv, respectively.

Recombinant vectors may be recovered from the above transformants usingknown methods (J. Sambrook et al., Molecular cloning, Vol. 1,1.25–1.28). For example, the cell membrane of a transformant may beweakened with solution 1 (50 mM glucose, 25 mM Tris.HCl, and 10 mMEDTA). With solution 2 (0.2N NaOH and 1% SDS) the cell membrane may bedestroyed and proteins and chromosomes may be denatured. The ingredientsother than recombinant vectors may be aggregated with solution 3 (5Mpotassium acetate and acetic acid) and then centrifuged. The obtainedrecombinant vector layer may be precipitated with ethanol to recoverrecombinant vectors.

The present invention includes, within its scope, a monoclonal antibodyvariable region, which consists of a light chain comprising the peptidesof SEQ ID Nos. 9, 10, and 11 and a heavy chain comprising the peptidesof SEQ ID Nos. 12, 13, and 14. Further, preferable is a monoclonalantibody variable region, wherein the light chain variable region hasthe amino acid sequence of SEQ ID NO. 7 and the heavy chain variableregion has the amino acid sequence of SEQ ID NO. 8.

From the above cDNA genes encoding a monoclonal antibody variable regionaccording to the present invention, a humanized monoclonal antibodyagainst HBV may be obtained by fusing the CDR region where S-surfaceantigen binds directly (i.e., in case of the light chain, the geneencoding the peptides of SEQ ID Nos. 9, 10, and 11; and in case of theheavy chain, the gene encoding the peptides of SEQ ID Nos. 12, 13, and14) to a human antibody gene, or by substituting a human antibodyvariable region with a gene encoding the monoclonal antibody variableregion according to the present invention.

As mentioned above, the gene encoding the monoclonal antibody variableregion according to the present invention is specifically effective inthe recognition of HBV S-surface antigen, especially determinant a,which has the highest expression ratio in the HBV surface antigens.Therefore, the gene according to the present invention may be used tomanufacture monoclonal antibodies which may be widely applied to varioustypes of HBV surface antigens, such as adr, adw, ayr and ayw, toneutralize and/or remove HBV.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be further illustrated in detail by, butis by no means limited to, the following Examples.

EXAMPLE 1 RNA Isolation from the Cell Line (A9-11-5) and its cDNASynthesis

After 1×108 of A9-11-5 cells were added to 10 ml of 4M guanidiniumthiocyanate to disrupt the cells, 8 ml of acidic phenol solution wasadded thereto. The mixture was centrifuged (10,000 rpm, 10 minutes) toextract the RNA. To 5 μg of the extracted RNA, were added 0.5 ng ofoligo d(T), 0.5 unit of RNase inhibitor and 100 unit of moloney murineleukemia virus reverse transcriptase (M-MLV). The resulting mixture wasreacted at 37° C. for 1 hour to synthesize cDNA.

Using 2 μg of the synthesized cDNA as a template; in case of the lightchain, the DNA oligomers of SEQ ID Nos. 1 and 2 as primers; and in caseof the heavy chain, the DNA oligomers of SEQ ID Nos. 3 and 4 as primers,polymerase chain reaction (PCR) was performed with the use of anAmpliTaq polymerase (Perkin-Elmer Biosystem Co., USA). In the first stepof the PCR, the reaction was repeated 30 cycles in the reactionconditions of 1.5 minute at 94° C., 2 minutes at 55° C., and 3 minutesat 72° C. In the second step, the reaction was performed 1 cycle in thecondition of 1.5 minute at 94° C., 2 minutes at 55° C., and 10 minutesat 72° C.

1.5% Agarose gel electrophoresis was performed using amplified PCRproduct. After stained with 100 ml of 0.5 μg/ml ethidium bromidesolution for 20 minutes, the amplified gene products appeared about 460bp in case of the heavy chain and about 440 bp in case of the lightchain compared to 100 bp of standard DNA ladder (Lifetechnology Co.USA).

EXAMPLE 2 cDNA Cloning

After removing impurities by adding 200 μl of phenol and 200 μl ofchloroform to the 440 bp gene fragment (the light chain gene fragment),which was recovered using a dialysis membrane (Spectrum Co. USA) afterperforming 1.5% agarose gel electrophoresis in Example 1, 2.5 ml ofethanol was added to purify the gene fragment. Purified gene fragmentwas subcloned into a pCRII vector (Invitrogen Co., USA) and E. coli DH5α(Lifetechnology Co., USA) was transformed therewith to give atransformant (Cohen, S. N. et al., Proc. Nat. Acad. Sci. 69, 2110,1972). The obtained transformant was cultured overnight in the LB mediumcontaining 100 μg/ml of ampicillin and, subsequently, processed to givea plasmid. Then, the plasmid was cut with a restriction enzyme EcoRI(Biolab Co., USA) to give clones Lv-1, Lv-4, and Lv-7 in which the above440 bp of gene fragment was inserted.

The same procedures were performed with the 460 bp gene fragment (theheavy chain gene fragment) to give a recombinant vector, with which E.coli DH5α (Lifetechnology Co., USA) was transformed to obtain atransformant. The transformant was cultured overnight in the LB mediumcontaining 100 μg/ml of ampicillin and subsequently, processed to give aplasmid. Then the plasmid was cut with a restriction enzyme EcoRI(Biolab Co., USA) to give clones Hv-1, Hv-4, and Hv-7 in which the above460 bp of gene fragment was inserted.

60 μl of polyethylene glycol solution (20% polyethylene glycol and 2.5MNaCl) was added to 100 ug/ml of each plasmid solution obtained from theabove clones and then centrifuged. 100 μl of distilled water was addedto the resulting precipitate, extracted twice with 50 μl of phenolsolution, and 200 μl of ethanol was used to purify plasmids.

5 μl of 2N sodium hydroxide and 100 μl of 10 mM EDTA were added to 50 μlof the solution containing 2 μg of the purified plasmid. Then, themixture was reacted at 37° C. for 30 minutes. To the reaction mixture, 1pmol of M13 and T7 primers were added, respectively. The whole mixturewas reacted 2 minutes at 65° C., and then allowed to stand to roomtemperature. The nucleotide sequences of each clone were analyzed usingDNA sequence version II kit (United States Biochemical Co., USA).

As a result, the nucleotide sequences of three light chain clones (Lv-1,Lv-4, and Lv-7) were identical. The plasmid vectors obtained from theseclones were named pCRA9Lv. And the transformants with pCRA9Lv plasmidvectors were named E. coli DH5α/YRC-pCRA9Lv which was originallydeposited in Korean Collection for Type Cultures (KCTC) in KoreaResearch Institute of Biosci. & Biotech on May 16, 2000 (KCTC 18021P)and then, converted to a deposit under the Budapest Treaty on May 16,2001 (KCTC 1011BP).

Further, the nucleotide sequences of three heavy chain clones (Hv-1,Hv-4, and Hv-7) were identical. The plasmid vectors obtained from theseclones were named pCRA9Hv. And the transformants with pCRA9Hv plasmidvectors were named E. coli DH5α/YRC-pCRA9Hv which was originallydeposited in Korean Collection for Type Cultures (KCTC) in KoreaResearch Institute of Biosci. & Biotech on May 16, 2000 (KCTC 18020P)and then, converted to a deposit under the Budapest Treaty on May 16,2001 (KCTC 1010BP).

EXAMPLE 3 Nucleotide Sequence Analysis of the cDNA

As a result of analysis on the variable region amino acid sequence(Harris. L. et al., Protein Sci. 4, 306–310, 1995.; Kabat. E. A. et al.,Sequence of proteins of immunological interest. 5th Ed., 1991.; WilliamsA. F. et al., Annu. Rev. Immunol. 6, 381–406, 1988) of the monoclonalantibody obtained from the cell line A9-11-5, it was identified that theheavy chain belongs to I(B) subgroup and the light chain belongs to λ1series.

Among the variable regions, the antigen-recognition CDR residues of theheavy chain were at the positions of 31–35 (CDR1), 50–65 (CDR2), and98–103 (CDR3) and those of the light chain were of 23–36 (CDR1), 52–58(CDR2), and 91–98 (CDR3).

EXAMPLE 4 Binding Affinity of the Monoclonal Antibody Obtained from theHybridoma Cell Line A9-11-5

2.0×10−11M of the monoclonal antibody obtained from the cell lineA9-11-5 was added to the solution of the HBV S-surface antigen(International Enzymes Inc., USA) at various concentrations(1.0×10−6˜1.0×10−12M) and then the mixture was reacted at roomtemperature for 3 hours.

100 μl of each mixture was added to 96-well immulon plates (Dinatech Co.USA) where 0.1 μg of above S-surface antigen was pre-coated. The mixturewas incubated 2 hours at 37° C. and the supernatant solution wasremoved. 200 μg of 0.5% casein-phosphate buffered saline was added toeach well and further incubated 1 hour at 37° C. 100 μl of the diluted(×1,000) goat anti-mouse polyclonal antibody to which horseradishperoxidase was conjugated (Sigma Co., USA) was added, and its opticaldensity was measured using ELISA reader (Dinatech Co., USA).

The monoclonal antibody obtained from the cell line A9-11-5 has highbinding affinity of 1.84×10−9 M-1. The term of binding affinity meansthat the reciprocal of the antigen concentration at which 50% ofmonoclonal antibody binding is inhibited (Friguet B. et al., J. ofImmunological Method, 77, 305–319, 1985)

While the invention has been described with respect to the specificembodiments, it should be recognized that various modifications andchanges may be made by those skilled in the art to the invention whichalso fall within the scope of the invention as defined as the appendedclaims.

1. A cDNA encoding a heavy chain variable region of a monoclonalantibody against S-surface antigen of hepatitis B virus, said heavychain variable region comprising the peptides of SEQ ID Nos. 12, 13, and14.
 2. The cDNA according to claim 1, wherein the heavy chain variableregion has the amino acid sequence of SEQ ID NO.
 8. 3. The cDNAaccording to claim 2, which comprises the nucleotide sequence of SEQ IDNO.
 6. 4. A recombinant vector pCRA9Hv comprising the cDNA of claim 1.5. A transformant E. coli DH5α/YRC-pCRA9Hv, which is transformed with arecombinant vector pCRA9Hv.